Efficient delivery of siRNA to specific cell populations in vivo remains a major challenge to successful therapeutic applications. Hematopoietic cells, including acute myeloid leukemia (AML) cells, pose special problem for siRNA delivery due to low transfection efficiency, which requires the use of lentiviral vectors. We recently demonstrated that ligands for intracellular receptors, such as TLR9, can be used as targeting moieties for cell-specific siRNA delivery. Novel dual-function CpG-siRNA conjugates, generated by synthetically linking siRNA to a CpG oligonucleotide (ODN), target and silence genes specifically in TLR9- positive immune cells including DCs, macrophages and B cells in mice. In contrast to the naked siRNA, the siRNA molecules equipped with a CpG moiety, are rapidly internalized by target cells and localized into endosomes in the absence of any transfection or packaging reagents. Our preliminary studies show that a new CpG-siRNA version, generated by utilizing the CpG sequence optimized for human cell stimulation, allows for gene targeting specifically in human TLR9-positive tumor cells, such as AML. We demonstrated that local as well as systemic administration of CpG-siRNAs targeting oncogenic and/or pro-survival genes induced tumor cell death and inhibited growth of xenotransplanted human AML tumors. To optimize CpG- siRNA strategy for use in anticancer therapy, we propose to define the molecular mechanisms and intracellular events involved in TLR9-mediated gene silencing. We also need to prolong stability and circulation half-life of CpG-siRNA reagents to maximize their efficacy for systemic administration. The nuclease-resistant and long-lived conjugates will be generated through chemical modifications and multimerization of CpG-siRNAs. Next, we plan to use the CpG-siRNA strategy to target STAT5, a transcription factor mediating survival of the vast majority of AML tumors. We will assess the effect of optimized CpG-STAT5 siRNA against disseminated AML tumor models and primary leukemic blasts from leukemia patients. Additionally, we will test the effect of CpG-STAT5 siRNA on viability and immune activation of normal human immune cells. Results from the proposed studies have potential to overcome major hurdles limiting the application of siRNA therapeutics, allowing for silencing of currently non- druggable target genes, like STAT5, thereby blocking AML proliferation and survival. We anticipate that with better understanding of the mechanism(s) underlying TLR9-mediated silencing effect and with CpG-siRNA conjugates optimized for use against disseminated tumors, the proposed studies will produce a technology platform applicable to broad clinical application against AML and other hematologic malignancies. In a long- term perspective, this work has potential to generate novel, more effective and safer therapeutics, expanding treatment options for the benefit of patients with various forms of blood cancers.